GSM6590589: OA3.Ts cells, mock-infected, 1; Ovis aries; RNA-Seq
Total RNA from each sample was extracted with TRIzol reagent (Invitrogen, USA). In order to construct mRNA library, poly(A) RNA was purified for two rounds from 2 μg total RNA using Dynabeads Oligo(dT)25-61005 (Thermo Fisher, USA). Approximately 2 μg of total RNA was used to remove ribosomal RNA according to the manuscript of the Ribo-Zero Gold Kit (Epicentre Biotechnologies, USA) to construct lncRNA or circRNA library. The poly(A) RNA and the remaining RNA after ribosomal RNA removal were fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, USA). The cleaved RNA fragments were reverse-transcribed into cDNA by SuperScript Ⅱ Reverse Transcriptase (Invitrogen), which were subsequently used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase Ⅰ (NEB), RNase H (NEB) and dUTP Solution (Thermo Fisher). An A-base was then added to the blunt ends of each strand, followed by ligation of indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPure XP beads (Beckman Coulter, USA). After the heat-labile UDG enzyme (NEB) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR. The average insert size for the final cDNA library was 300±50 bp.